POS0436 EFFECT OF DISEASE-MODIFYING ANTI-RHEUMATIC DRUGS ON LUNG MICROENVIRONMENT IN RHEUMATOID ARTHRITIS-ASSOCIATED LUNG DISEASE ANIMAL MODELS
Authors: S. H. Chang, J. S. Lee, J. J. Chae, J. Y. Choe, E. Y. Lee
Journal: Annals of the Rheumatic Diseases (2022)

Background Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is one of the pivotal extrapulmonary conditions. However, the pathophysiology of RA-ILD, including the effect of disease-modifying anti-rheumatoid drugs (DMARDs) is largely unknown. Objectives To study the effect of methotrexate and TNFα inhibitor on lung microenvironment of SKG mice using single-nucleus RNA sequencing. Methods At age eight to ten weeks, male SKG mice received a single intraperitoneal zymosan A (ZyA) injection to induce arthritis and pneumonitis. We gave them twice-weekly intraperitoneal injections of PBS, methotrexate (7.5 mg/kg; MilliporeSigma), or TNFα inhibitor (100 μg/kg; R&amp;D Systems, Inc., Minneapolis, MN, USA). Nuclei were prepared from frozen lung tissue under ribonuclease-free conditions by a method adapted from an existing protocol. Results Macroscopically, pneumonitis was most evident in SKG mice with ZyA and methotrexate treatment; multiple inflammatory cell aggregations were observed. In mice with ZyA and TNFα inhibitor, there were a few perivascular inflammatory cell aggregations as in the mice with ZyA and PBS, compared with the mice without ZyA. A total of 59,860 nuclei were obtained from sixteen mice, four mice from each of the four groups. We classified each cell type using previous reports on the single-nucleus cells of mouse lungs. The most frequently observed cell was the type 2 alveolar (AT2) cell among all four groups. Alveolar epithelial cells were further subclustered into six clusters (Figure 1). Cluster 2 highly expressed type 1 alveolar (AT1) cell marker genes. In mice without ZyA, cluster 0 predominated relative to cluster 1. This proportion was inverted in mice with the ZyA injection, where cluster 1 predominated. TNFα inhibitor treatment reversed the proportion, with cluster 0 predominating in this group. On the other hand, methotrexate treatment further accentuated the predominance of cluster 1 over cluster 0. Interestingly, a distinct cluster 3 was observed after methotrexate treatment. This distinctive alveolar epithelial cell cluster (cluster 3) showed IL-1, TNF, IFNα, and IFNγ perturbation-response signatures using Library of Integrated Network-Based Cellular Signatures (LINCS) 1000 ligand perturbation analysis. In addition, enrichment analysis revealed that cluster 3, as well as cluster 2 (i.e., AT1 cells) significantly enriched genes of BMI1 knockout mice that fail to self-renew their lung cells after damage. Conclusion The current study shows that methotrexate exacerbates lung inflammation via attenuation of the regenerative potential of AT2 cells. References [1]Sakaguchi N, et al. Nature 2003;426:454. [2]Koenitzer JR, et al. Am J Respir Cell Mol Biol 2020;63:739. [3]Dovey JS, et al. Proc Natl Acad Sci USA 2008;105:11857. Acknowledgements This work was supported by the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare, Republic of Korea (grant no.HI14C1277). Disclosure of Interests None declared.